Cyclic di-GMP (c-di-GMP) is a near-ubiquitous, newly appreciated second messenger signal in bacteria that contributes to pathogenicity-promoting behaviors including biofilm formation, motility, virulence factor expression, development, and quorum sensing. Its signaling pathways are thus potentially attractive targets for new approaches to combat biofilm-based or acute infections, but the mechanisms by which it regulates transcription in bacteria are largely unknown. The goal of our research is to elucidate, and thus potentially enable therapeutic targeting of, the mechanisms that mediate c-di-GMP signaling in bacteria by integrating genetic, biochemical, chemical, structural, bioinformatic, and computational approaches. We and others have previously found that a subset of transcription factors belonging to the NtrC-like bacterial enhancer binding protein (EBP) family directly bind and respond to c-di-GMP. EBPs are widespread in bacteria, and regulate fundamental bacterial behaviors including biofilm formation, motility, quorum sensing, and virulence factor expression. We further found that c-di-GMP binds to and inhibits the ability of the Vibrio cholerae ?54- dependent EBP FlrA to induce motility. Our preliminary data suggest that c-di-GMP inhibits transcription by locking dimeric FlrA into a conformation incapable of DNA binding, but conversely binds to and activates the ?70-dependent V. cholerae EBP VpsR to induce biofilm formation. We hypothesize that c-di-GMP activates transcription by stimulating VpsR oligomerization. In Aims 1 & 2 we will test these hypotheses, using combined in vivo and in vitro genetic and biochemical assays to identify critical structural determinants for this regulation and define the impact of c-di-GMP on transcription factor activity. These studies will be integrated with the elucidation of the X-ray crystal structures of FlrA and VpsR in the presence and absence of c-di-GMP binding to formulate a mechanistic model of c-di-GMP regulation of EBPs. Elucidating these mechanisms will allow us to identify among the thousands of EBPs in diverse bacterial species those that are c-di-GMP-regulated. Preliminary studies generating crystals of purified FlrA proteins, and the identification of c-di-GMP-insensitive, constitutively active FlrA and VpsR mutants support the feasibility of these studies. In Aim 3 we will expand our analysis to identify novel c-di-GMP-dependent transcriptional machinery in V. cholerae and completely define the c-di-GMP-dependent regulatory network. This analysis will fully harness newly developed deep sequencing technologies (TN-seq, RNA-seq, and IPODHR). We will use these data to formulate a computation model of the c-di-GMP regulon in V. cholerae, gaining an appreciation for the global impact of c-di-GMP on this pathogen and uncovering fundamental principles that generally underpin c-di-GMP regulatory networks. Our studies will advance current concepts of the control of bacterial transcriptional initiation, identifying novel targets or development of new antibiotics that are agonists or antagonists of c-di-GMP-mediated regulation in pathogenic bacterial species.